Haemophilus Influenzae Type IV PILI

ABSTRACT

The invention described herein relates to a  Haemophilus influenzae  ( H. influenzae ) regulon encoding type IV pili. In particular, the invention relates to type IV pili from nontypeable  H. influenzae  (NTHi) and from  H. influenzae  strains a, b, c, e and f. The invention provides isolated  H. influenzae  pilus polynucleotides and polypeptides encoded by the polynucleotides as well as polynucleotides and polypeptides encoded by the polynucleotides involved in the assembly/disassembly of the structure. The invention also relates to uses of these polynucleotides and/or polypeptides including methods for eliciting an immune response to  H. influenzae  and methods of treating and preventing  H. influenzae  related pathological conditions.

Experimental work relating to the invention described herein wassupported by grants R01 DCO3915 and R01 DC005980 from the NIH/NIDCD. TheUnited States government may have certain rights in the invention.

FIELD OF INVENTION

The invention described herein relates to a Haemophilus influenzae (H.influenzae) regulon encoding type IV pili. In particular, the inventionrelates to type IV pili from nontypeable H. influenzae (NTHi) and fromH. influenzae strains a, b, c, e and f. The invention provides isolatedH. influenzae pilus polynucleotides and polypeptides encoded by thepolynucleotides as well as polynucleotides and polypeptides encoded bythe polynucleotides involved in the assembly/disassembly of thestructure. The invention also relates to uses of these polynucleotidesand/or polypeptides including methods for eliciting an immune responseto H. influenzae and methods of treating and preventing H. influenzaerelated pathological conditions.

BACKGROUND

The clinical term for middle ear infections is otitis media (OM).According to Klein, Vaccine, 19 (Suppl. 1): S2-S8, 2000, OM is the mostcommon reason for an ill child to obtain healthcare and for a child inthe United States to receive antibiotics or undergo a generalanesthetic. Statistics indicate that 24.5 million physician officevisits were made for OM in 1990, representing a greater than 200%increase over those reported in the 1980s. While rarely associated withmortality, the morbidity associated with OM is significant. Hearing lossis a common problem associated with this disease, often affecting achild's behavior, education and development of language skills (Baldwin,Am. J. Otol., 14: 601-604, 1993; Hunter et al., Ann. Otol. Rhinol.Laryngol. Suppl., 163: 59-61, 1994; Teele et al., J. Infect. Dis., 162:685-694, 1990). The socioeconomic impact of OM is also great, withdirect and indirect costs of diagnosing and managing OM exceeding $5billion annually in the U.S. alone (Kaplan et al., Pediatr. Infect. Dis.J., 16: S9-11, 1997).

OM is thought to result from infectious, environmental and host geneticsfactors. Bacteria such as Haemophilus influenzae, Streptococcuspneumoniae and Moraxella catarrhalis are the most common infectiousorganisms in OM. Acute OM is a disease characterized by rapid onset andshort duration of signs and symptoms of inflammation in the middle ear,while chronic OM refers to a condition that is defined by the relativelyasymptomatic presence of fluid (or effusion) in the middle ear. However,in chronic OM, despite the absence of certain signs of acute infection(i.e., ear pain or fever), these abnormal middle ear fluids can persistfor periods exceeding three months. Treatment of acute OM by antibiotictherapy is common, but antibiotic-resistant bacteria have emerged.Surgical management of chronic OM involves the insertion of tympanostomytubes through the tympanic membrane of the ear while a child is undergeneral anesthesia. While this procedure is commonplace (prevalencerates are ˜13%; Bright et al., Am. J. Public Health, 83(7): 1026-8,1993) and is highly effective in terms of relieving painful symptoms bydraining the middle ear of accumulated fluids, it is invasive andcarries incumbent risks (Berman et al., Pediatrics, 93(3):353-63, 1994;Bright et al., supra.; Cimons, ASM News, 60: 527-528; Paap, Ann.Pharmacother., 30(11): 1291-7, 1996). There is thus a need foradditional approaches to the management and, preferably, the preventionof OM.

OM vaccine development is most advanced for S. pneumoniae, the primarycausative agent of acute OM (AOM), as evidenced by the recent approvaland release of a seven-valent capsular-conjugate vaccine, PREVNAR®(Eskola and Kilpi, Pedriatr. Infect. Dis. J. 16: S72-78, 2000). WhilePREVNAR® has been highly efficacious for invasive pneumococcal disease,coverage for OM has been disappointing (6-8%) with reports of anincreased number of OM cases due to serotypes not included in thevaccine (Black et al., Pedriatr. Infect. Dis J, 19: 187-195, 2000;Eskola et al., Pedriatr. Infect. Dis J., 19: S72-78, 2000; Eskola etal., N Engl. J. Med., 344: 403-409, 2001; Snow et al., Otol. Neurotol.,23: 1-2, 2002).

H. influenzae is a gram-negative bacterium that, as noted above, plays arole in OM. Clinical isolates of H. influenzae are classified either asserotypes “a” through “f” or as non-typeable depending on the presenceor absence, respectively, of type-specific polysaccharide capsules onthe bacteria. A vaccine for H. influenzae type b has been developed.Like Prevnar®, the type b H. influenzae vaccines target thepolysaccharide capsule of this organism and thus the vaccine iscomprised of capsule polysaccharide that has been conjugated to aprotein carrier. Less progress has been made for a vaccine fornon-typeable H. influenzae (NTHi) which causes approximately 20% ofacute OM in children and predominates in chronic OM with effusion(Coleman et al., Inf and Immunity, 59(5), 1716-1722, 1991; Klein,Pedriatr. Infect. Dis J., 16, S5-8, 1997; Spinola et al., J. Infect.Dis., 154, 100-109, 1986). NTHi can also cause pneumonia, sinusitis,septicemia, endocarditis, epiglottitis, septic arthritis, meningitis,postpartum and neonatal infections, postpartum and neonatal sepsis,acute and chromic salpingitis, epiglottis, pericardis, cellulitis,osteomyelitis, endocarditis, cholecystitis, intraabdominal infections,urinary tract infection, mastoiditis, aortic graft infection,conjunctitivitis, Brazilian purpuric fever, occult bacteremia andexacerbation of underlying lung diseases such as chronic bronchitis,bronchietasis and cystic fibrosis. A prototype NTHi isolate is the lowpassage isolate 86-028NP which was recovered from a child with chronicOM. This strain has been well characterized in vitro (Bakaletz et al.,Infect. Immun., 53: 331-5, 1988; Holmes et al., Microb. Pathog., 23:157-66, 1997) as well as in a chinchilla OM model (Bakaletz et al.,Vaccine, 15: 955-61, 1997; Suzuki et al., Infect. Immun., 62: 1710-8,1994; DeMaria et al., Infect. Immun., 64: 5187-92, 1996). The NTHistrain 86-026NP was deposited with the American Type Culture Collection,10801 University Blvd., Manassas, Va. 20110, on Oct. 16, 2001 andassigned accession no. PTA-4764. A contig set from the genome of stain86-028NP can be found at http://www.microbial-pathogenesis.org.

Adherence and colonization are acknowledged first steps in thepathogenesis of H. influenzae. As such, H. influenzae express multipleadhesins including hemagglutinating pili, fimbriae and non-fimbrialadhesins (Gilsdorf et al., Pediatr Res 39, 343-348, 1996; Gilsdorf.,Infect. Immun., 65, 2997-3002, 1997; and St. Geme III, Cell. Microbiol.,4, 191-200, 2002). Notably, none of the adhesins described havepreviously been associated with a motility function. Moreover, H.influenzae do not express flagella with are also associated withmotility. Twitching motility is a flagella-independent fog in ofbacterial translocation over moist surfaces and occurs by extension,tethering, and then retraction of polar structures known as type IV pili(Bardy., Microbiology, 149, 295-304, 2003; Tonjum and Koomey, Gene, 192,155-163, 1997; Wolfgang et al., EMBO J., 19, 6408-6418; Mattick, Annu.Rev. Microbiol., 56, 289-314, 2002). Type IV pili are typically 5-7 nmin diameter, several micrometers in length and comprised of a singleprotein subunit assembled into a helical conformation with 5 subunitsper turn (Bardy et al., Microbiology, 149, 295-304, 2003; Wall andKaiser, Mol. Microbiol., 32, 1-10, 1999). Type IV pilin subunits areusually 145-160 amino acids in length and may be glycosylated orphosphorylated. There are two classes of pilin subunits, type IVa andtype IVb, which are distinguished from one another by the average lengthof the leader peptide and the mature subunit, which N-methylated aminoacid occupies the N-terminal position of the mature protein, and theaverage length of the D-region (for disulfide region). Most of therespiratory pathogens express class IVa pilins, whereas theenteropathogens more typically express class IVb pilins. Type IVa piliare distinguished by the presence of a highly conserved, hydrophobicN-terminal methylated phenylalanine.

Type IV pili serve as a means of rapid colonization of new surfaces.Thus type IV pilus expression is important to both adherence and biofilmformation by many bacteria (Mattick, Annu. Rev. Microbiol., 56, 289-3142002; O'Toole and Kolter, Mol. Microbiol., 30, 295-304, 1998; Klausen etal., Mol. Microbiol., 50, 61-68, 2003; Jesaitis et al., J. Immunol.,171, 4329-4339, 2003), as well as virulence of Neisseria species,Moraxella bovis, Vibrio cholerae, enteropathogenic Escherichia coli andPseudomonas aeruginosa, among others (O'Toole and Kolter, supra; Klausenet al., supra; Klausen et al., Mol. Microbiol., 48, 1511-1524, 2003;Strom and Lory, Annu. Rev. Microbiol., 47, 565-596, 1993). A biofilm isa complex organization of bacteria that are anchored to a surface via abacterially extruded exopolysaccharide matrix. The matrix envelopes thebacteria and protects it from the human immune system. Ehrlich et al.,JAMA, 287(13), 1710-1715 (2002) describes biofilm formation by H.influenzae. It has been postulated that blocking the interaction betweentype IV pili and the human body can avoid or stop the bacterialinfection (Meyer et al., U.S. Pat. No. 6,268,171 issued Jul. 31, 2001).

Type IV pilus expression is a complex and highly regulated bacterialfunction. In P. aeruginosa, the biogenesis and function of type IV piliis controlled by over forty genes (Strom and Lory, supra). To date, onlya subset of the vast number of related type IV pilus genes (Tonjum andKoomey, supra; Darzins and Russell, Gene, 192, 109-115, 1997) have beenfound in several members of the HAP (Haemophilus, Actinobacillus andPasteurella) family (Stevenson et al., Vet. Microbiol., 92, 121-134,2003; Doughty et al., Vet. Microbiol., 72, 79-90, 2000; Dougherty andSmith, Microbiology, 145, 401-409 1999), but neither expression of typeIV pili nor twitching motility has ever been described for any H.influenzae isolate. In fact, H. influenzae is classically described as abacterium that does not express these structures (Friedrich et al. Appl.Environ. Microbiol., 69, 3695-3700, 2003; Fussenegger et al., Gene, 192,125-134, 1997), despite the presence of a cryptic gene cluster withinthe strain Rd genome (Fleischmann et al., Science, 269, 496-512, 1995).Strain Rd is a non-encapsulated derivative of an H. influenzae serotyped organism (Zwahlen et al., Infect. Immun., 42, 708-715, 1983; Bendlerand Goodgal, J. Microbial., 70, 411-422, 1972; Risberg et al., Eur. J.Biochem., 261, 171-180, 1999). Although strain Rd has some virulenceproperties, serotype d strains are generally considered to becommensals; they do not frequently cause disease (Dames et al., J. Med.Microbiol., 52, 277-282, 2003). It is therefore important to make thedistinction between disease-causing strains of H. influenzae and strainRd.

SUMMARY OF THE INVENTION

The present invention relates to Type IV pilus gene clusters of H.influenzae, in particular non-typeable H. influenzae (NTHi) and H.influenzae strains a, b, c, e and f.

Polynucleotides and Polypeptides of the Invention

The present invention provides H. influenzae polynucleotides andparticularly open reading frames from a regulon arranged in two geneclusters plus one other gene. The regulon includes a gene (pilA) thatencodes the major subunit of a heretofore uncharacterized H. influenzaetype IV pilus. The regulon includes polynucleotides from a gene clusterencoding pilin polypeptides PilA (major pilin subunit), PilD (leaderpeptidase), PilB and PilC (involved in the assembly/disassembly of thepilin structure); another gene cluster encoding ComA, ComB, ComC, ComD,ComE, and ComF (involved in competence for transformation and pilusexpression); and a gene encoding PilF (required for type IV pilusbiogenesis) (Watson et al, Gene, 49: 56, 1996). In one embodiment, thepilus regulon is that of NTHi H. influenzae strain 86-028NP.

Polynucleotides encoding the NTHi 86-028NP pilin polypeptides set out inthe following SEQ ID NOs are provided by the invention: PilA polypeptidein SEQ ID NO: 2, PilB polypeptide in SEQ ID NO: 4, PilC polypeptide inSEQ ID NO: 6, PilD polypeptide in SEQ ID NO: 8, ComA polypeptide in SEQID NO: 10, ComB polypeptide in SEQ ID NO: 12, ComC polypeptide in SEQ IDNO: 14, ComD polypeptide in SEQ ID NO: 16, ComE polypeptide in SEQ IDNO: 18, ComF polypeptide in SEQ ID NO: 20 and PilF polypeptide in SEQ IDNO: 22. Alternative codon usage is thus specifically contemplated by theinvention. In one embodiment, the polynucleotides comprise the NTHi86-028NP gene sequences set out in the following SEQ ID NOs whichrespectively encode the foregoing polypeptides: pilA in SEQ ID NO: 1,pilB in SEQ ID NO: 3, pilC in SEQ ID NO: 5, pilD in SEQ ID NO: 7, comAin SEQ ID NO: 9, comB in SEQ ID NO: 11, comC in SEQ ID NO: 13, comD inSEQ ID NO: 15, comE in SEQ ID NO: 17, comF in SEQ ID NO: 19; and pilF inSEQ ID NO: 21. Each of the polynucleotide sequences includes a finalthree nucleotides representing a stop codon.

Also provided are polynucleotides encoding PilA polypeptides from NTHiclinical isolates 1728MEE, 1729MEE, 3224A, 10548MEE, 1060MEE, 1885MEE,1714MEE, 1236MEE, 1128MEE and 214NP. The amino acid sequences of thesePilA polypeptides are set out in SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38,40, 42 and 44, respectively. Again, the possibility of alternative codonusage is specifically contemplated in polynucleotides encoding thepolypeptides. In one embodiment, the polypeptides are respectivelyencoded by the nucleotide sequences set out in SEQ ID NOs: 25, 27, 29,31, 33, 35, 37, 39, 41 and 43.

The invention provides for polynucleotides that hybridize understringent conditions to (a) the complement of the nucleotide sequencesset out in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27,29, 31, 33, 35, 37, 39, 41 or 43; (b) a polynucleotide which is anallelic variant of any polynucleotides recited above; (c) apolynucleotide which encodes a species homolog of any of the proteinsrecited above; or (d) a polynucleotide that encodes a polypeptidecomprising a specific domain or truncation of the polypeptides of thepresent invention. Type IV pilin polynucleotides from other non-typeableH. influenzae strains and from H. influenzae strains a, b, c, e and fare specifically contemplated. These polynucleotides can be identifiedand isolated by techniques standard in the art such as hybridization andpolymerase chain reaction using part or all of the polynucleotides ofSEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33,35, 37, 39, 41 or 43 as probes or primers, respectively.

The polynucleotides of the invention also include nucleotide sequencesthat are substantially equivalent to the polynucleotides recited above.Polynucleotides according to the invention can have, e.g., at least 65%,at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% andeven more typically at least 95%, 96%, 97%, 98% or 99% sequence identityto the NTHi polynucleotides recited above.

Included within the scope of the nucleic acid sequences of the inventionare nucleic acid sequence fragments that hybridize under stringentconditions to the NTHi nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, orcomplements thereof, which fragment is greater than about 5 nucleotides,preferably 7 nucleotides, more preferably greater than 9 nucleotides andmost preferably greater than 17 nucleotides. Fragments of, e.g., 15, 17,or 20 nucleotides or more that are selective for (i.e., specificallyhybridize to any one of the polynucleotides of the invention) arecontemplated. These nucleic acid sequence fragments capable ofspecifically hybridizing to a NTHi polynucleotide of the invention canbe used as probes to detect NTHi polynucleotides of the invention and/orcan differentiate NTHi polynucleotides of the invention from otherbacterial genes, and are preferably based on unique nucleotidesequences.

The term “stringent” is used herein to refer to conditions that arecommonly understood in the art as stringent. Hybridization stringency isprincipally determined by temperature, ionic strength, and theconcentration of denaturing agents such as formamide. Examples ofstringent conditions for hybridization and washing are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodiumchloride, 0.0015M sodium citrate, and 50% formamide at 42° C. SeeSambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.,Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989).

More stringent conditions (such as higher temperature, lower ionicstrength, higher formamide, or other denaturing agent) may also be used,however, the rate of hybridization will be affected. In instanceswherein hybridization of deoxyoligonucleotides is concerned, additionalexemplary stringent hybridization conditions include washing in 6×SSC0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-baseoligos).

Other agents may be included in the hybridization and washing buffersfor the purpose of reducing non-specific and/or backgroundhybridization. Examples are 0.1% bovine serum albumin, 0.1%polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodiumdodecylsulfate, NaDodSO₄, (SDS), ficoll, Denhardt's solution, sonicatcdsalmon sperm DNA (or other non-complementary DNA), and dextran sulfate,although other suitable agents can also be used. The concentration andtypes of these additives can be changed without substantially affectingthe stringency of the hybridization conditions. Hybridizationexperiments are usually carried out at pH 6.8-7.4, however, at typicalionic strength conditions, the rate of hybridization is nearlyindependent of pH. See Anderson et al., Nucleic Acid Hybridisation: APractical Approach, Ch. 4, IRL Press Limited (Oxford, England).Hybridization conditions can be adjusted by one skilled in the art inorder to accommodate these variables and allow DNAs of differentsequence relatedness to form hybrids.

As noted above, polynucleotides contemplated by the present inventionare not limited to the specific polynucleotides of SEQ ID NOs: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43,but also include, for example, allelic and species variations thereof.Allelic and species variations can be routinely determined by comparingthe sequence provided in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, preferably the openreading frames therein, a representative fragment thereof, or anucleotide sequence at least 90% identical, preferably 95% identical, tothe open reading frames within SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43 with a sequencefrom another isolate of the same species or another species. Preferredcomputer program methods to determine identity and similarity betweentwo sequences include, but are not limited to, the GCG program package,including GAP (Devereux et al., Nucl. Acid. Res., 12: 387, 1984;Genetics Computer Group, University of Wisconsin, Madison, Wis.),BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215: 403-410,1990). The BLASTX program is publicly available from the National Centerfor Biotechnology Information (NCBI) and other sources (BLAST Manual,Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al.,supra). The well known Smith-Waterman algorithm may also be used todetermine identity.

Polynucleotides of the invention may be isolated from natural sources ormay be synthesized by standard chemical techniques, e.g., thephosphotriester method described in Matteucci et al., J Am Chem. Soc.,103: 3185 (1981).

Antisense polynucleotides complementary to the polynucleotides encodingthe pilus polypeptides of the invention are also provided.

Polypeptides of the invention include pilin polypeptides PilA, PilD,PilB, PilC, ComA, ComB, ComC, ComD, ComE, ComF and PilF. In oneembodiment the polypeptides comprise the NTHi 86-028NP amino acidsequences respectively set out in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20 or 22. Polypeptides of the invention also include PilApolypeptides set out in SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42or 44. In additional embodiments, the Type IV pilin polypeptides of theinvention are those of other non-typeable H. influenzae strains and fromH. influenzae strains a, b, c, e and f.

Polypeptides of the invention specifically include peptide fragments(i.e., peptides) that retain one or more biological or immunogenicproperties of a full length polypeptide of the invention. In oneembodiment PilA peptide fragments provided by the invention aredesignated TfpQ2, TFPQ3, TfpQ4 and OLP3 and respectively comprise aminoacids 35 through 68 of SEQ ID NO: 2, amino acids 69 through 102 of SEQID NO: 2, amino acids 103 through 137 of SEQ ID NO: 2, and amino acids21 through 35 of SEQ ID NO: 2.

The invention also provides for polypeptides with one or moreconservative amino acid substitutions that do not affect the biologicaland/or immunogenic activity of the polypeptide. Alternatively, thepolypeptides of the invention are contemplated to have conservativeamino acids substitutions which may or may not alter biologicalactivity. The term “conservative amino acid substitution” refers to asubstitution of a native amino acid residue with a normative residue,including naturally occurring and nonnaturally occurring amino acids,such that there is little or no effect on the polarity or charge of theamino acid residue at that position. For example, a conservativesubstitution results from the replacement of a non-polar residue in apolypeptide with any other non-polar residue. Further, any nativeresidue in the polypeptide may also be substituted with alanine,according to the methods of “alanine scanning mutagenesis”. Naturallyoccurring amino acids are characterized based on their side chains asfollows: basic: arginine, lysine, histidine; acidic: glutamic acid,aspartic acid; uncharged polar: glutamine, asparagine, serine,threonine, tyrosine; and non-polar: phenylalanine, tryptophan, cysteine,glycine, alanine, valine, proline, methionine, leucine, norleucine,isoleucine General rules for amino acid substitutions are set forth inTable 1 below.

TABLE 1 Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asn GlyPro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, LeuLeu Norleucine, Ile, Val, Met, Leu Lys Arg, 1,4 Diaminobutyric Arg MetLeu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Arg Pro Ala Gly Ser Thr,Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe ValIle, Met, Leu, Phe, Ala, Leu

The invention also provides variants of the polypeptides of the presentinvention (e.g., a polypeptide exhibiting at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically atleast about 95%, 96%, 97%, more typically at least about 98%, or mosttypically at least about 99% amino acid identity to a polypeptide of SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22) that retain biologicaland/or immunogenic activity.

The invention contemplates that polynucleotides of the invention may beinserted in a vector for amplification or expression. For expression,the polynucleotides are operatively linked to appropriate expressioncontrol sequences such as promoter and polyadenylation signal sequences.Further provided are host cells comprising polynucleotides of theinvention. Exemplary prokaryotic host cells include bacteria such as E.coli, Bacillus, Streptomyces, Pseudomonas, Salmonella and Serratia.Methods of producing polypeptides of the invention by growing the hostcells and isolating polypeptide from the host cells or growth medium arespecifically contemplated. One or more polynucleotides from the pilusregulon may be expressed in a host cell. For example, expression of thepilA gene alone and expression of multiple polynucleotides from thepilus regulon in order to affect assembly of the native pili structureare both specifically contemplated. Alternatively, polypeptides of theinvention can be prepared by chemical synthesis using standard means.Particularly convenient are solid phase techniques (see, e.g., Eriksonet al., The Proteins (1976) v. 2, Academic Press, New York, p. 255).Automated solid phase synthesizers are commercially available. Inaddition, modifications in the sequence are easily made by substitution,addition or omission of appropriate residues. For example, a cysteineresidue may be added at the carboxy teiminus to provide a sulfhydrylgroup for convenient linkage to a carrier protein, or spacer elements,such as an additional glycine residue, may be incorporated into thesequence between the linking amino acid at the C-terminus and theremainder of the peptide.

The term “isolated” refers to a substance removed from, and essentiallyfree of, the other components of the environment in which it naturallyexists. For example, a polypeptide is separated from other cellularproteins or a DNA is separated from other DNA flanking it in a genome inwhich it naturally occurs.

Antibodies

The invention provides antibodies which bind to antigenic epitopesunique to (i.e., are specific for) H. influenzae pilus polypeptides ofthe invention. Also provided are antibodies which bind to antigenicepitopes common among multiple H. influenzae subtypes but unique withrespect to any other antigenic epitopes. The antibodies may bepolyclonal antibodies, monoclonal antibodies, antibody fragments whichretain their ability to bind their unique epitope (e.g., Fv, Fab andF(ab)2 fragments), single chain antibodies and human or humanizedantibodies. Antibodies may be generated by techniques standard in theart using pilun polypeptide(s) of the invention or host cells expressingpilin polypeptide(s) of the invention as antigens.

The present invention provides for antibodies specific for the pilinpolypeptides of the present invention and fragments thereof, whichexhibit the ability to kill both H. influenzae bacteria and to protecthumans from infection. The present invention also provides forantibodies specific for the polypeptides of the invention which reducethe virulence, inhibit adherence, inhibit biofilm formation, inhibittwitching motility, inhibit cell division, and/or inhibit penetrationinto the epithelium of H. influenzae bacteria and/or enhancephagocytosis of the H. influenzae bacteria.

In vitro complement mediated bactericidal assay systems (Musher et al.,Infect. Immun. 39: 297-304, 1983; Anderson et al., J. Clin. Invest. 51:31-38, 1972) may be used to measure the bactericidal activity ofanti-pilus antibodies.

It is also possible to confer short-term protection to a host by passiveimmunotherapy via the administration of pre-formed antibody against anH. influenzae polypeptide of the invention. Thus, antibodies of theinvention may be used in passive immunotherapy. Human immunoglobulin ispreferred in human medicine because a heterologous immunoglobulin mayprovoke an immune response to its foreign immunogenic components. Suchpassive immunization could be used on an emergency basis for immediateprotection of unimmunized individuals subject to special risks.

In another embodiment, antibodies of the invention may be used in theproduction of anti-idiotypic antibody, which in turn can be used as anantigen to stimulate an immune response against pilin epitopes.

Methods for Eliciting an Immune Response and Compositions Therefor

The invention contemplates methods of eliciting in an individual animmune response to one or more H. influenzae type IV pilus polypeptides.In certain embodiments, the methods elicit an immune response to thePilA protein. These methods elicit one or more immune responses,including but not limited to, immune responses which inhibit bacterialreplication, immune responses which block H. influenzae adherence tocells, immune responses which prevent H. influenzae twitching and immuneresponses which prevent biofilm formation. In one embodiment, themethods comprise a step of administering an immunogenic dose of acomposition comprising one or more polypeptides of the invention. Inanother embodiment, the methods comprise administering an immunogenicdose of a composition comprising a cell expressing one or morepolypeptides of the invention. In yet another embodiment, the methodscomprise administering an immunogenic dose of a composition comprisingone or more polynucleotides encoding one or more polypeptides of theinvention. The polynucleotide may be a naked polynucleotide notassociated with any other nucleic acid or may be in a vector such as aplasmid or viral vector (e.g., adeno-associated virus vector oradenovirus vector). The methods may be used in combination in a singleindividual. The methods may be used prior or subsequent to H. influenzaeinfection of an individual.

In one embodiment of methods of the invention, a composition of theinvention is administered as a priming dose followed by one or morebooster doses. Co-administration of proteins or polypeptides thatbeneficially enhance the immune response such as cytokines (e.g., IL-2,IL-12, GM-CSF), cytokine-inducing molecules (e.g. Leaf) or costimulatorymolecules is also contemplated.

An “immunogenic dose” of a composition of the invention is one thatgenerates, after administration, a detectable humoral (antibody) and/orcellular (T cell) immune response in comparison to the immune responsedetectable before administration or in comparison to a standard immuneresponse before administration. The invention contemplates that theimmune response resulting from the methods may be protective and/ortherapeutic. In a preferred embodiment, the antibody and/or T cellimmune response protects the individual from H. influenzae infection,particularly infection of the middle ear and/or the nasopharynx or lowerairway. In this use, the precise dose depends on the patient's state ofhealth and weight, the mode of administration, the nature of theformulation, etc., but generally ranges from about 1.0 μg to about 5000μg per 70 kilogram patient, more commonly from about 10 to about 500 μgper 70 kg of body weight.

Humoral immune response may be measured by many well known methods, suchas Single Radial Immunodiffussion Assay (SRID), Enzyme Immunoassay (EIA)and Hemagglutination Inhibition Assay (HAI). In particular, SRIDutilizes a layer of a gel, such as agarose, containing the immunogenbeing tested. A well is cut in the gel and the serum being tested isplaced in the well. Diffusion of the antibody out into the gel leads tothe formation of a precipitation ring whose area is proportional to theconcentration of the antibody in the serum being tested. EIA, also knownas ELISA (Enzyme Linked Immunoassay), is used to determine totalantibodies in the sample. The immunogen is adsorbed to the surface of amicrotiter plate. The test serum is exposed to the plate followed by anenzyme linked immunoglobulin, such as IgG. The enzyme activity adherentto the plate is quantified by any convenient means such asspectrophotometry and is proportional to the concentration of antibodydirected against the immunogen present in the test sample. HAI utilizesthe capability of an immunogen such as viral proteins to agglutinatechicken red blood cells (or the like). The assay detects neutralizingantibodies, i.e., those antibodies able to inhibit hemagglutination.Dilutions of the test serum are incubated with a standard concentrationof immunogen, followed by the addition of the red blood cells. Thepresence of neutralizing antibodies will inhibit the agglutination ofthe red blood cells by the immunogen. Tests to measure cellular immuneresponse include determination of delayed-type hypersensitivity ormeasuring the proliferative response of lymphocytes to target immunogen.

The invention correspondingly provides compositions suitable foreliciting an immune response to pilus polypeptides of the invention. Asnoted above, the compositions comprise one or more pilus polypeptides,cells expressing one or more polypeptides, or one or morepolynucleotides encoding one or more pilus polypeptides. Thecompositions may also comprise other ingredients such as carriers andadjuvants.

In compositions of the invention, a pilus polypeptide may be fused toanother protein when produced by recombinant methods. In one embodiment,the other protein may not, by itself, elicit antibodies, but itstabilizes the first protein and forms a fusion protein retainingimmunogenic activity. In another embodiment, the fusion proteincomprises another protein that is immunogenic, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilize the fusion protein and facilitateproduction and purification thereof. The other protein may act as anadjuvant in the sense of providing a generalized stimulation of theimmune system. The other protein may be fused to either the amino orcarboxy terminus of the NTHi protein of the invention.

In other compositions of the invention, pilus polypeptides may beotherwise linked to carrier substances. Any method of creating suchlinkages known in the art may be used. Linkages can be formed withheterobifunctional agents that generate a disulfide link at onefunctional group end and a peptide link at the other, such as adisulfide amide forming agent, e.g., N-succidimidyl-3-(2-pyridyldithio)proprionate (SPDP) (See, e.g., Jansen et al., Immun. Rev. 62:185, 1982)and bifunctional coupling agents that form a thioether rather than adisulfide linkage such as reactive esters of 6-maleimidocaproic acid,2-bromoacetic acid, 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid and the like, andcoupling agent which activate carboxyl groups by combining them withsuccinimide or 1-hydroxy-2-nitro-4-sulfonic acid, for sodium salt suchas succinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC).

The pilus polypeptides may be formulated as neutral or salt forms.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the peptide) and which are formedwith inorganic acids such as, e.g., hydrochloric or phosphoric acids, orsuch organic acids as acetic, oxalic, tartaric, mandelic. Salts formedwith the free carboxyl groups may also be derived from inorganic basessuch as, e.g., sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,2-ethylamino ethanol, histidine, and procaine.

Compositions of the invention may further comprise adjuvants. Knownadjuvants include, for example, emulsions such as Freund's Adjuvants andother oil emulsions, Bordetella pertussis, MF59, purified saponin fromQuillaja saponaria (QS21), aluminum salts such as hydroxide, phosphateand alum, calcium phosphate, (and other metal salts), gels such asaluminum hydroxide salts, mycobacterial products including muramyldipeptides, solid materials, particles such as liposomes and virosomes.Examples of natural and bacterial products known to be used as adjuvantsinclude monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-likeacylated monosaccharide), OM-174 which is a lipid A derivative from E.coli, holotoxins such as cholera toxin (CT) or one of its derivatives,pertussis toxin (PT) and heat-labile toxin (LT) of E. coli or one of itsderivatives, and CpG oligonucleotides. Adjuvant activity can be affectedby a number of factors, such as carrier effect, depot formation, alteredlymphocyte recirculation, stimulation of T-lymphocytes, directstimulation of B-lymphocytes and stimulation of macrophages.

Compositions of the invention are typically formulated as injectables,either as liquid solutions or suspensions; solid farms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. The preparation may also be emulsified. The active immunogenicingredient is often mixed with excipients, which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, e.g., water, saline, dextrose, glycerol, ethanol, or thelike and combinations thereof. In addition, if desired, the vaccine maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, or adjuvants, which enhance theeffectiveness of the vaccine. The vaccines are conventionallyadministered parenterally, by injection, for example, eithersubcutaneously or intramuscularly.

Additional formulations which are suitable for other modes ofadministration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers mayinclude, for example, polyalkalene glycols or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1-2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25-70%.

Compositions may also be administered through transdermal routesutilizing jet injectors, microneedles, electroporation, sonoporation,microencapsulation, polymers or liposomes, transmucosal routes andintranasal routes using nebulizers, aerosols and nasal sprays.Microencapsulation using natural or synthetic polymers such as starch,alginate and chitosan, D-poly L-lactate (PLA), D-polyDL-lactic-coglycolic microspheres, polycaprolactones, polyorthoesters,polyanhydrides and polyphosphazenes polyphosphatazanes are useful forboth transdermal and transmucosal administration. Polymeric complexescomprising synthetic poly-ornithate, poly-lysine and poly-arginine oramphipathic peptides are useful for transdermal delivery systems. Inaddition, due to their amphipathic nature, liposomes are contemplatedfor transdermal, transmucosal and intranasal vaccine delivery systems.Common lipids used for vaccine delivery includeN-(1)2,3-(dioleyl-dihydroxypropyl)-N,N,N,-trimethylammonium-methylsulfate (DOTAP), dioleyloxy-propyl-trimethylammonium chloride DOTMA,dimystyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE),dimethyldioctadecyl ammonium bromide (DDAB) and9N(N′,N-dimethylaminocthane) carbamoyl) cholesterol (DC-Chol). Thecombination of helper lipids and liposomes will enhance up-take of theliposomes through the skin. These helper lipids include, dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine(DLPE), dimyristoyl phosphatidylethanolamine (DMPE),dipalmitoylphosphatidylethanolamine (DPPE). In addition, triterpenoidglycosides or saponins derived from the Chilean soap tree bark (Quillajasaponaria) and chitosan (deacetylated chitan) have been contemplated asuseful adjuvants for intranasal and transmucosal vaccine delivery.

Formulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use.

Methods of Inhibiting H. influenzae

Alternatively, the invention includes methods of inhibiting H.influenzae type IV pili function in an individual. The methods compriseadministering to the individual, for example, one or more antibodies ofthe invention; one or more polypeptides of the invention; one or moreantisense polynucleotides of the invention; one or more RNAi molecules;and/or one or more small molecules, in an amount that inhibits functionof the pili. In vitro assays may be used to demonstrate the ability toinhibit pili function. Embodiments of these methods include, forexample, methods using inhibitors of pilus polypeptide synthesis and/orpilus assembly, inhibitors of adherence mediated via type IV pili,inhibitors that disrupt existing biofilms mediated by type IV pili, andinhibitors of twitching.

Inhibition is contemplated for any pathological condition involving H.influenzae, for example, OM, pneumonia, sinusitis, septicemia,endocarditis, epiglottitis, septic arthritis, meningitis, postpartum andneonatal infections, postpartum and neonatal sepsis, acute and chromicsalpingitis, epiglottis, pericardis, cellulitis, osteomyelitis,endocarditis, cholecystitis, intraabdominal infections, urinary tractinfection, mastoiditis, aortic graft infection, conjunctitivitis,Brazilian purpuric fever, occult bacteremia and exacerbation ofunderlying lung diseases such as chronic bronchitis, bronchietasis andcystic fibrosis.

Compositions comprising inhibitors of H. influenzae type IV pilifunction are provided. The compositions may consist of one of theforegoing active ingredients alone, may comprise combinations of theforegoing active ingredients or may comprise additional activeingredients used to treat bacterial infections. As discussed above, thecompositions may comprise one or more additional ingredients such aspharmaceutically effective carriers. Also as discussed above, dosage andfrequency of the administration of the compositions are determined bystandard techniques and depend, for example, on the weight and age ofthe individual, the route of administration, and the severity ofsymptoms. Administration of the pharmaceutical compositions may be byroutes standard in the art, for example, parenteral, intravenous, oral,buccal, nasal, pulmonary, rectal, intranasal, or vaginal.

Animal Model

Methods of the invention may be demonstrated in a chinchilla modelwidely accepted as an experimental model for OM. In particular, achinchilla model of NTHi-induced OM has been well characterized(Bakaletz et al., J. Infect. Dis., 168: 865-872, 1993; Bakaletz andHolmes, Clin. Diagn. Lab. Immunol., 4: 223-225, 1997; Suzuki andBakaletz, Infect. Immun., 62: 1710-1718, 1994; Mason et al., Infect.Immun., 71:3454-3462, 2003), and has been used to determine theprotective efficacy of several NTHi outer membrane proteins,combinations of outer membrane proteins, chimeric synthetic peptidevaccine components, and adjuvant formulations against OM (Bakaletz etal., Vaccine, 15: 955-961, 1997; Bakaletz et al., Infect. Immun., 67:2746-2762, 1999; Kennedy et al., Infect. Immun., 68: 2756-2765, 2000;Kyd et al., Infect. Immun., 66:2272-2278, 2003; Novotny and Bakaletz, J.Immunol., 171, 1978-1983, 2003).

In the model, adenovirus predisposes chinchillas to H.influenzae-induced OM media, which allowed for the establishment ofrelevant cell, tissue and organ culture systems for the biologicalassessment of NTHi (Bakaletz et al., J. Infect. Dis., 168: 865-72, 1993;Suzuki et al., Infect. Immunity 62: 1710-8, 1994). Adenovirus infectionalone has been used to assess the transudation of induced serumantibodies into the tympanum (Bakaletz et al., Clin. Diagnostic LabImmunol., 4(2): 223-5, 1997) and has been used as a co-pathogen withNTHi, to determine the protective efficacy of several active and passiveimmunization regimens targeting various NTHi outer membrane proteins,combinations of OMPs, chimeric synthetic peptide vaccine components, andadjuvant formulations as vaccinogens against otitis media (Bakaletz etal., Infect Immunity, 67(6): 2746-62, 1999; Kennedy et al., Infect.Immun., 68(5): 2756-65, 2000; Novotny et al., Infect Immunity 68(4):2119-28, 2000; Poolman et al., Vaccine 19 (Suppl. 1): S108-15, 2000).

Methods of Detecting H. influenzae Bacteria

Also provided by the invention are methods for detecting bacteria in anindividual. In one embodiment, the methods comprise detecting pilipolynucleotides of the invention in a biological sample using primers orprobes that specifically bind to the polynucleotides. Detection of thepolynucleotide may be accomplished by numerous techniques routine in theart involving, for example, hybridization and/or PCR. In anotherembodiment, the methods comprise detecting pili polypeptides of theinvention in a biological sample using antibodies of the invention thatspecifically bind to the polypeptides. The antibodies may be used in anyimmunoassay system known in the art including, but not limited to,radioimmunoassays, ELISA assays, sandwich assays, precipitin reactions,gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, fluorescent immunoassays, protein A immunoassaysand immunoelectrophoresis assays. Biological samples to be utilized inthe methods include, but are not limited to, blood, serum, ear fluid,spinal fluid, sputum, urine, lymphatic fluid and cerebrospinal fluid.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an alignment of amino acid sequences of the PilA polypeptidesof NTHi Rd, 86-028NP (SEQ ID NO: 2), 1728MEE (SEQ ID NO: 26), 1729MEE(SEQ ID NO: 28), 3224A (SEQ ID NO: 30), 10548MEE (SEQ ID NO: 32),1060MEE (SEQ ID NO: 34), 1885MEE (SEQ ID NO: 36), 1714MEE (SEQ ID NO:38), 1236MEE (SEQ ID NO: 40), 1128MEE (SEQ ID NO: 42), 214NP (SEQ ID NO:44).

DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrate the invention wherein Example 1describes sequences of NTHi strain 86-028NP type IV pilus genes of theinvention and detection of the pilA gene in thirteen clinical H.influenzae isolates, Example 2 demonstrates classical type IVpilus-dependent aggregate formation by NTHi strain 86-028NP, Example 3demonstrates twitching motility in NTHi strain 86-028NP, Example 4describes observation of type IV pili on NTHi strain 86-928NP bynegative staining and transmission electron microscopy, Example 5describes the generation of a pilA mutant, Example 6 describesexperiments in a chinchilla model of infection with NTHi and pilA mutantNTHi, Example 7 describes pilA genes from ten NTHi clinical isolates,Example 8 describes experiments demonstrating an immune response to NTHipilli in children and Example 9 describes the identification of NTHipeptide fragments for use as immunogens.

Example 1

A type IV pilus regulon was identified in an OM isolate of NTHi.

Many strains of NTHi, including strain 86-028NP, do not possess the hiflocus required for the expression of hemagglutinating LKP pili(Mhlanga-Mutangadura et al., J. Bacteriol., 180(17), 4693-4703, 1988),yet most form biofilms. Because pili are important to biofilm formationin other bacterial systems, the contig set from the NTHi strain 86-028NPgenomic sequencing effort (see http://www.microbial-pathogenesis.org orco-owned U.S. Ser. No. 60/453,134) was analyzed for genes potentiallyencoding another type of pilus. The dataset was BLASTed using the tblasnalgorithm with default parameters using the Pseudomonas aeruginosaproteins annotated as related to the type IV pilus or twitching motilityfor P. aeruginosa including PilQ and PilT at www.Pseudomonas.com. Thetranslated polypeptide for the P. multocida PilA protein was also usedin this search (Doughty et al., Vet. Microbial., 72, 79-90, 2000).

What initially appeared to be a cryptic type IV pilus gene locus wasidentified. Specifically, in strain 86-028NP, there are four genes thatare highly homologous to those in H. influenzae strain Rd, A.pleuropneumoniae and P. multocida (Stevenson et al., Vet. Microbiol.,92, 121-134, 2003; Doughty et al., supra; Zhang et al., FEMS MicrobiolLett, 189, 15-18, 2000; and Ruffolo et al., Infect. Immun., 65, 339-343,1997). These genes encode PilA, PilB, PilC and PilD in strain Rd(Dougherty and Smith, Microbiology, 145(2), 401-409, 1999).

The NTHi strain 86-028NP regulon includes a gene cluster ofpolynucleotides encoding pilin polypeptides PilA (major pilin subunit),PilD (leader peptidase), PilB and PilC (contemplated to be transcribedfrom the same mRNA and to be involved in the assembly/disassembly of thepilin structure); a gene cluster of polynucleotides encoding pilinpolypeptides ComA, ComB, ComC, ComD, ComE, and ComF (involved incompetence for transformation and pilus expression); and another geneencoding PilF (required for type IV pilus biogenesis). The amino acidsequences of the pilin polypeptides set out in the following SEQ ID NOs:PilA in SEQ ID NO: 2, PilB in SEQ ID NO: 4, PilC in SEQ ID NO: 6, PilDin SEQ ID NO: 8, ComA in SEQ ID NO: 10, ComB in SEQ ID NO: 12, ComC inSEQ ID NO: 14, ComD in SEQ ID NO: 16, ComE in SEQ ID NO: 18, ComF in SEQID NO: 20 and PiIF in SEQ ID NO: 22. The gene sequences encoding thepolypeptides are set out in the following SEQ ID NOs which respectivelyencode the foregoing polypeptides: pilA in SEQ ID NO: 1, pilB in SEQ IDNO: 3, pilC in SEQ ID NO: 5, pilD in SEQ ID NO: 7, comA in SEQ ID NO: 9,comB in SEQ ID NO: 11, comC in SEQ ID NO: 13, comD in SEQ ID NO: 15,comE in SEQ ID NO: 17, comE in SEQ ID NO: 19; and pilF in SEQ ID NO: 21.Each of the polynucleotides sequences includes a final three nucleotidesrepresenting a stop codon.

In gene expression profiling studies employing cDNA microarrays tocharacterize the regulation of NTHi genes during the development ofcompetence (i.e., the natural ability of NTHi to take up foreign DNApotentially enhancing or expanding its genetic diversity), the corngenes as well as the pil genes are up-regulated during competencedevelopment.

In a Southern blot experiment using pilA sequences as a probe, thirteenlow-passage clinical NTHi OM isolates recovered from patients undergoingtympanostomy and tube insertion for chronic otitis media and oneclinical isolate recovered from a patient with cystic fibrosis had asingle copy of pilA within their genome. These fourteen total isolateswere designated by the following strain numbers, respectively: 86-028NP;1728MEE; 1729MEE; 1714MEE; 214NP; 1236MEE; 165NP; 1060MEE; 1128MEE;10548MEE; 3224A; 3185A, 1885MEE and 27W11679INI.

In the experiment, bacterial chromosomal DNA was isolated using aPUREGENE DNA isolation kit from Gentra Systems (Minneapolis, Minn.),digested with MfeI and the digests run on a 0.8% agarose gel. DNA wastransferred to a Nytran SuPerCharge membrane using the Turbo Blotter kit(Schleicher & Schuell, Keene, N.H.). The probe was generated by PCRamplification of the coding sequence of the 86-028NP pilA gene using theprimers 5′ tgtgacacttccgcaaaaa (SEQ ID NO: 23) and 5′taataaaaggaaaatgaatga (SEQ ID NO: 24). The amplicon was purified using aQIAquick PCR purification kit (Qiagen Inc., Valencia, Calif.). Followingthe manufacturer's directions, 100 ng of purified PCR product waslabeled with horseradish peroxidase using the ECL Direct Nucleic AcidLabeling and Detection System (Amersham Biosciences UK Ltd., LittleChalfont, Bucks, UK). Developed blots were exposed to Fuji Super RxX-ray film (Fuji Photo Film Co., Tokyo, Japan).

The PilA polypeptide of NTHi strain 86-028NP has a predicted andapparent molecular mass of approximately 14 kDa, contains an N-terminalmethylated phenylalanine.

Example 2

NTHi strain 86-028NP formed classic aggregates in a sub-agar surfacetranslocation assay and a surface growth assay when grown underconditions of nutrient depletion.

NTHi strain 86-028NP was grown on chocolate agar for 18-20 hours (37°C., 5% CO₂, in a humidified atmosphere) prior to all experiments.Subsequently, this organism was inoculated onto either sBHI, a richmedium on which the NTHi grows very well, or a chemically defined mediumwhich supports the growth of H. influenzae (Coleman et al. J. Clin.Micro., 41:4408-4410, 2003) that comprised 83% RPMI1640 media (GibcoBRL, Rockville, Md.), sodium pyruvate (87.3 mM) (Gibco BRL), β-NAD(0.0087 mg/ml) (Sigma Chemical Co., St Louis, Mo.), HEME-histidine(0.0175 mg/ml) (Sigma), Uracil (0.087 mg/ml) (Sigma), and inosine (1.75mg/ml) (Sigma).

Both agars were poured into one of two formats, in sterile 8-wellchamber slides (Lab-tech, Naperville, Ill.) or into sterile 35 mm glasspetri dishes (Fisher Scientific, location). When the glass slide wasseparated from the 8-well chamber slides, the agar remained within thechambers, thus enabling use of the “bottom” surface of the agar forinoculation, which is optimal for assay of twitching motility due to therelative smoothness of this surface (Semmler et al., Microbiology, 145,2863-2873, 1999 and Mattick, Ann. Rev. Microbiol., 56, 289-314, 2002).Whereas agars cast into 8-well chamber slides were used to demonstrate asurface growth phenotype, agars poured into the petri dishes were usedfor demonstration of sub-surface agar translocation (Semmler et al.,supra) whereas agars cast into 8-well chamber slides were used todemonstrate agar surface growth phenotype. All assays were repeated aminimum of three times, on separate days.

Agars that had been poured into sterile glass petri dishes wereinoculated subsurface with 0.5 μl of a suspension of NTHi grown asdescribed above, using a sterile micropipet tip. Plates were observedafter 24 hours incubation (37° C., 5% CO₂) and were then held at roomtemperature (25° C.) for an additional 24 hours prior to re-reading forsigns of bacterial translocation between the bottom surface of the agarand the glass petri dish.

On sBHI medium, 24 hours post-inoculation, NTHi was observed to havegrown in a small area (˜0.5 mm radius) surrounding the inoculation sitebetween the agar and the glass petri dish bottom. After an additional 24hours, the growth pattern remained similar to that observed at 24 hours.On the chemically defined medium after 24 hours of incubation, growth ofNTHi was observed between the agar surface and the glass petri dishbottom, at a distance 2 to 5 mm from the inoculation site. The bacteriahad also aggregated into small colonies in a halo-like patternsurrounding the inoculation site. After 48 hours, NTHi had formed a verydistinct array of micro-colonies with many occurring at a distance>5 mmfrom the inoculation site. The formation of micro-satellites up to 5 mmdistance from original site of inoculation was a hallmark finding ofgrowth on chemically defined medium and was never seen when strain86-028NP was grown on rich agar.

Chamber slides were either inoculated with 0.5 ul of a suspension of18-20 hour chocolate agar-grown NTHi [suspended in sterile pyrogen freesaline (American Pharmaceutical Partners Inc., Schaumburg, Ill.)], or asingle colony was stabbed for transfer to the surface of the agar with asterile toothpick.

On sBHI medium, thirty minutes post-inoculation, NTHi appeared in closeassociation with the agar surface and was growing in a sheet-likepattern. At 2.5 hours, approximately 80-90% of the surface area wascovered with a thin sheet of bacteria. Also at this time,micro-aggregates of NTHi began to appear. At 6-7 hours post-inoculation,these micro-aggregates were discernable with the naked eye and therewere approximately 3-5 micro-aggregates per well. In addition, NTHi werestill observed to be growing as a sheet that covered approximately50-70% of the agar surface. Twenty-four hours after inoculation, NTHiappeared as large single colonies at each inoculation site.

On chemically defined medium, like that observed on sBHI, thirty minutespost-inoculation, NTHi appeared to be growing in sheet, however thedensity of the bacteria appeared much less than that observed on sBHIagar. At 2.5 hours after inoculation, numerous micro-aggregates wereevident throughout the agar surface. In contrast to those noted whenNTHi was inoculated onto sBHI agar, these micro-aggregates were largerand much more dense in appearance. Approximately 30-40 micro-aggregatescould be seen on each well. There was still a large area of sheet likegrowth of NTHi at this time point, with approximately 80% of the surfacearea covered by bacteria. At 6-7 hours post inoculation, themicro-aggregates were larger, denser, and easily seen with the nakedeye. Also, areas of radial growth or halos were seen radiating outwardfrom large colonies, similar to the growth patterns described forNeisseria and Pseudomonas sp. (refs). By this time period, most of thebacteria appeared to be arranged in small clusters or micro-aggregateswith a very small proportion seen covering the agar surface as a sheetor monolayer. After 24 hours, there were large single colonies at eachinoculation site, however there were also many small satellite coloniespresent over the entire agar surface, including sites remote from thepoints of inoculation.

Thus, NTHi strain 86-028NP demonstrates classic aggregate formation,similar to that reported for type IV pilus-expressing P. aeruginosa(Semmler et al., Microbiology, 145(10), 2863-2873, 1999), when grownunder conditions of nutrient depletion.

Example 3

The movement of individual NTHi cells between a glass coverslip and asmooth agar surface was traced by video-microscopy. The cells moved atapproximately 0.42 μm/sec, consistent with that reported for twitchingP. aeruginosa (Skerker and Berg, Proc. Natl. Acad., Sci. USA, 98,6901-6904, 2001) and Neisseria gonorrhoeae (Merz et al., Nature, 207,98-102, 2000).

A loopful of NTHi stain 86-028NP, grown on chocolate agar at 37° C. and5% CO₂ for 20 hours and then held at ambient temperature for anadditional 24 hours, was suspended in sterile water and 0.5 μl of theresulting suspension was placed onto a sterile glass slide. To providecontrast and thus aid visualization, 0.5 μl of trypan blue (0.4%, Sigma,St. Louis Mo.) was added to the bacterial suspension. The droplet wasthen covered with a sterile coverslip and viewed via light microscope(Axioskope 40, Zeiss, Thornwood, N.Y.). Specimens were observed at roomtemperature over a period of approximately 15-20 minutes. The bacteriawere readily observable and directional movement of several, althoughnot all, cells or microaggregates of cells was noted. In order tostimulate activity, we added 0.5 μl of a heme solution (1 mg/ml) (Sigma,St. Louis, Mo.) to one side of the sterile coverslip. Twitching activitywas documented by the capture of both video [video otoscopy system(MEDR_(X) Inc, Seminole, Fla.) attached to a VCR] and still images inorder to determine length and rate of excursions.

Individual cells, or microaggregates of cells, traveled a total lineardistance of approximately 11.0 μm over a period of 51 seconds (rateapproximately 0.22 μm/sec). However, over the entire period ofobservation, the rate of twitching motility observed ranged from 0.14 to0.48 μm/sec.

Example 4

Type IV pili were visualized by negative staining and transmissionelectron microscopy.

Overnight cultures of NTHi strain 86-028NP were inoculated onto sBHI anddefined agar plates and incubated for 2, 6 or 24 hours at 37° C., 5%CO₂. Additionally, cultures were inoculated into sBHI broth and definedbroth and incubated for 2.5 or 5.5 hours. These latter time pointsrepresent entry into exponential and lag phases of growth, respectively.Bacteria were then negatively stained using a Whatman-filtered solutioncontaining 2.0% ammonium acetate w/v (Sigma) and 2.0% ammonium molybdatew/v (Sigma) in sterile water (Bakaletz et al. Infect Immun, 198856:331-5). Foimvar- and carbon-coated copper grids, 300 mesh, (ElectronMicroscopy Sciences) were touched to individual colonies grown on agarplates, and then floated on a droplet of the negative stain solution.Broth-grown cultures were pelleted, the bacteria resuspended in sterilewater and grids were floated on equal volumes of bacterial suspensionand negative stain. After 5 minutes, grids were blotted and allowed toair dry prior to viewing in an Hitachi Model H-600 transmission electronmicroscope with attached video monitor (Gatan, Inc., Pleasanton, Calif.)and digital imaging system (Gatan, Inc.).

When NTHi were grown on sBHI, no type IV pilus-like structures wereobserved. Conversely, when grown under defined nutrient conditions, NTHiwas seen to express structures of approximately 6-7 nm diameter. Many ofthese structures were also found free on the grid surface. There wereapproximately 5 to 6 pili per bacterial cell and these were polar inlocation.

Example 5

A mutant deficient in the expression of PilA was generated to furthercharacterize components of the structures observed when strain 86-028NPwas grown in alkaline conditions on chemically defined media.

The pilA gene and approximately 1 kb 5′ and 3′ of the gene from strain86-028NP was amplified by PCR, cloned into pGEM-T Easy (Promega) and theDNA sequence determined to verify that there were no changes in thesequence in the clone as a result of the PCR amplification. As there wasno convenient restriction site in the pilA gene, a BamHI site wasengineered into the gene using the Stratagene QuikChange Site-DirectedMutagenesis Kit. The resulting construct was linearized with BamHI andthe gene was insertionally inactivated with the ΩKn-2 cassette(Perez-Casal et al., J Bacteriol., 173: 2617-2624, 1991). The resultingconstruct was linearized and transformed into strain 86-028NP using theMIV method (Poje and Redfield, p. 57-70 in Herbert et al. (Eds.),Haemophilus influenzae Protocols, Humana Press Inc., Toronto, 2003).Kanamycin-resistant clones were selected and insertional inactivation ofthe 86-028NP pilA gene was verified in selected clones by Southernhybridization.

When the pilA mutant was evaluated for expression of type IV pili aftergrowth under conditions that induced the increased expression of type IVpili in the parental isolate (Example 4), no cell-associated or freetype IV pili were observed confirming that the pilA gene product (and/orthe pilBCD gene products since the mutation is pilA is likely to disruptthe downstream gene products) are required for pilus expression.

Example 6

To determine whether type IV pili are necessary for colonization of thenasopharynx, as well as survival in and/or ability to form a biofilm inthe middle ear, we challenged fourteen adult chinchillas bothintranasally and transbullarly with either the parent strain 86-028NP orwith an isogenic pilA mutant (Example 5). On days 2, 5, 10, 15 and 20post-challenge, nasopharyngeal lavages and epitympanic taps wereperformed, and both nasal and middle ear mucosae were retrieved from 1-2chinchillas per cohort to determine cfu of NTHi in each of theseanatomic sites. Both the parent and pilA mutant were able to survive inthe chinchilla host for twenty days. However, whereas both strains werepresent in equivalent amounts in lavage and tap fluids, when assayed foran adherent subpopulation in tissue homogenates of nasal mucosae, thepilA mutant was absent from, or below our ability to detect, in 80% ofthe homogenates recovered after day 5 whereas 87% of similar nasalmucosae recovered from animals challenged with the parental isolate wereculture positive.

Confocal microscopy was performed on snap-frozen tissue to determinewhether a biofilm was present. The biomass formed by the pilA mutant inthe middle ear was of a different character than the well-structuredbiofilm characteristic of the parental isolate. The data indicate thatNTHi type IV pili play a key role in the disease course of OM.

Example 7

The pilA gene of ten clinical isolates of NTHi have been sequenced. Thenucleotide and amino acid sequences from the isolates are respectivelyset out as follows: 1728MEE in SEQ ID NOs: 25 and 26, 1729MEE in SEQ IDNOs: 27 and 28, 3224A in SEQ ID NOs: 29 and 30, 10548MEE in SEQ ID NOs:31 and 32, 1060MEE in SEQ ID NOs: 33 and 34, 1885MEE in SEQ ID NOs: 35and 36, 1714MEE in SEQ ID NOs: 37 and 38, 1236MEE in SEQ ID NOs: 39 and40, 1128MEE in SEQ ID NOs: 41 and 42, and 214NP in SEQ ID NOs: 43 and44. An alignment of the amino acid sequences with those of the pilApolypeptides from Rd and 86-028NP is presented in FIG. 1.

The pilA genes of all isolates encode a 12-residue leader peptide thatis largely invariant save a Q to L substitution at position 6 in twoisolates as well as in strain Rd. Mature PilA contains 137 residues andis predicted to contain a characteristic methylated phenylalanine atposition +1. Tyrosine residues at positions +24 and +27, and believed tobe involved in subunit-subunit interactions, are highly conserved as arefour Cys residues at positions +50, +60, +119 and +132. Interestingly,the NTHi PilA proteins appear to represent a new class of type IV pili.The leader peptide is larger than that characteristic for type IVapilins (typically 5-6 residues in length), yet shorter than the typicalIVb leader peptide (15-30 residues). At 137 residues, the mature NTHipilin is shorter than either class IVa or IVb pilins (150 and 190residues, respectively). Since the NTHi PilA proteins begin with anN-methylated phenylalanine, they are more like class IVa pilins howeverin electron micrographs, free NTHi type IV pili always appear inlaterally associated bundles, a phenotype more classically associatedwith class IVb pilins due to their ability to self-associate throughanti-parallel interactions.

In terms of NTHi PilA sequence diversity, overall these sequences arehighly homologous. See FIG. 1. Two areas of potentially importantdiversity, if surface accessible and also targeted for vaccinedevelopment due to protective immunodominance or adhesin-bindingfunction, exist at positions 55-64 and 79-87. Within the first region,amongst the clinical isolates, there appears to be two major variants,one representing the majority (seven of eleven isolates, 64%) andcharacterized by the following sequence: NET/ITNCT/MGGK and the otherrepresenting the minority (four of eleven isolates, 36%) andcharacterized by the sequence: GKP/LST/SCSGGS. There are however someadditional minor variations at positions +57 and +61 in the majoritygrouping and at positions +57 and +59 for the minority grouping. Thediversity noted at position +61 is only seen in one isolate to date(strain #1885), wherein there is a T to M substitution. Within thesecond focused region of diversity (position 79-87), there appears to betwo equally distributed variants among the clinical NTHi isolates. Thesequence ASVKTQSGG is present in five of eleven clinical isolates(˜45%), whereas the sequence KSVTTSNGA is present in six of elevenclinical isolates (˜55%).

Overall, of the seven isolates with the majority sequence at position55-64, five isolates also have the KSVTTSNGA motif at region 79-87, withthe remaining two isolates having the ASVKTQSGG motif in this region. Ofthe four remaining clinical isolates with the minority sequence atposition 55-64, three of these also have the ASVKTQSGG motif at region79-87, with only one isolate having the KSVTTSNGA sequence in thisdomain. Thereby, depending on whether or not these are conservativesubstitutions or not and if the sequences reside within surfaceaccessible, hydrophilic areas of high antigenic index and thus aretargets for vaccine development, they may or may not need to be includedas type IV pili-based components for inducing an immune response toNTHi.

Example 8

To examine the role of type IV pili in NTHi-induced OM and determine ifantibodies from children during natural disease recognize type IV pili,four sequential synthetic peptides were synthesized representing aminoacids 21-137 of SEQ ID NO: 2 of the mature PilA of NTHi strain 86-028NPand assayed via biosensor versus a panel of pediatric sera and middleear effusions obtained from children with OM. Serum from children at 2,6-7, or 18-19 mos and 4-6 yrs of age with OM due to NTHi were segregatedinto low and high incidence groups, as deteiinined by the number ofepisodes of OM.

To date, antibodies in sera obtained from children 2 and 6-7 mos of ageof either high or low incidence of OM demonstrated limited reactivitywith any of the type IV pili peptides, with values of 3-38 and 4-61resonance units (RU), respectively. However, a striking differencebetween these groups was seen with serum obtained at 18-19 mos of age.Whereas values obtained with sera from the low incidence 18-19 mos groupwere 44-105 RU, sera from the high incidence panel recognized the typeIV pili peptides up to five-fold greater (81-528 RU). At 4-6 yrs of age,as children naturally resolve OM, reactivity to the type IV pilipeptides was again similar between the two incidence groups. To confirmthat the reactivity observed here was specific for disease due to NTHi,sera from children with OM due to S. pneumoniae were also assayed. Inall cases, RU values of 16-120 were obtained versus all type IV pilipeptides. To assay for the presence of antibodies directed against typeIV pili in effusions obtained from the middle ears, we also assayedthese fluids via biosensor. Whereas effusions from children with OM dueto Streptococcus were unreactive, those recovered from children with OMdue to NTHi were highly reactive with type IV pili peptides.Collectively, the data strongly suggests that NTHi type IV pili areexpressed in vivo, during the disease course of OM and that thesestructures are immunogenic.

Example 9

Identification of immunogens that confer broad cross-protective immuneresponses against NTHi may be carried out as follows.

Synthesis of NTHi Pilin Peptides

In order to map both immunodominant and adhesin-binding domains of PilA,a panel of overlapping sequential peptides as well as peptides derivedfrom two focused areas of known diversity (see Example 6 above) aresynthesized. For example, thirteen 15-mer peptides with a 5-residueoverlap will be synthesized to map the entire 137 residue mature pilinprotein. The final C-terminal peptide will actually be a 17-mer spanningresidues 121-137 in order to incorporate the final two amino acids ofmature PilA. To accommodate the two described regions of diversity, twovariants of the peptide that spans residues 51-65 and two variants ofthe peptide that spans residues 79-95 will be synthesized. In order tofully accommodate this latter region of diversity, two peptides are madevarying in length by one amino acid at the N-terminus since the regionof diversity actually spans residues 79-87. Due to the additionalresidue, each of these latter two peptides will be 16-mers in length.Thus a total of fifteen peptides will be synthesized: twelve will be15-mers, one will be a 17-mer and two will be 16-mer peptides. Thepeptides are set out in Table 2 below wherein amino acid residue numberscorrespond to amino acids in SEQ ID No: 2.

TABLE 2 Peptide Sequence OLP1 [Residues 1-15] FTLIELMIVIAIIAIOLP2 [Residues 11-25] AIIAILATIAIPSYQ OLP3[Residues 21-35]IPSYQNYTKKAAVSE OLP4 [Residues 31-45] AAVSELLQASAPYKAOLP5 [Residues 41-55] APYKADVELCVYSTN OLP6vA [Residues 51-65]VYSTNETTNCTGGKN OLP6vB [Residues 51-65] VYSTGKPSTCSGGSNOLP7 [Residues 61-75] TGGKNGIAADITTAK OLP8 [Residues 71-85]ITTAKGYVKSVTTSN OLP9vA [Residues 79-94]77-95 YVKSVTTSNGAITVKGDGTOLP9vB [Residues 79 -94]77-95 YVASVKTQSGGITVKGNGTOLP10 [Residues 91-105] KGDGTLANMEYILQA OLP11 [Residues 101-115]YILQATGNAATGVTW OLP12 [Residues 111-125] TGVTWTTTCKGTDASOLP13 [Residues 121-137] GTDASLFPANFCGSVTQGeneration of Recombinant NTHi Pilin (rPilA)

Recombinant PilA protein (rPilA) may be generated to serve as a morereadily renewable product for use in assays to represent the entirepilin subunit protein. To do this, the published protocol of Keizer etal. (J. Biol. Chem., 276: 24186-14193, 2001), who studied a pilin whichalso had four Cys residues as it will be critical that rPilA similarlybe properly folded so as to possess functional qualities of the nativepilin subunit, is utilized. Briefly, a truncated pilin is engineeredwherein the first 28 residues are removed from the N-terminus to preventaggregation, and this truncated pilin will be further engineered to betransported to the periplasm by means of the incorporation of an OmpAleader sequence in the construct. Using this strategy Keizer et al.generated a recombinant soluble monomeric P. aeruginosa pilin proteinthat was able to bind to its receptor (asialo GM1) in in vitro assaysand decrease morbidity and mortality in mice when the peptide wasdelivered 15 mins. prior to heterologous challenge. This soluble,monomeric, truncated form of NTHi PilA will be useful in the studiesdescribed below.

Mapping Immunodominant Domains of PilA

The peptides and native and recombinant PilA proteins are used inconcert with both acute and convalescent chinchilla and pediatric serain addition to middle ear fluids from chinchillas and childrenexperiencing either experimental or natural OM due to NTHi (allavailable within our current specimen collection or planned forcollection as part of a separate initiative) to map immunodominantdomains of PilA via ELISA and also biosensor assays. Briefly, PilApeptides, rPilA and native pili are bound to 96-well microtiter platesor to a biosensor chip surface, then assayed for the relative amount ofantibody within serum or middle ear fluid samples that binds to eachpeptide.

These studies identify those regions of the pilin subunit that arerelatively more immunodominant than others as recognized by both thechinchilla host and the human child. Due to the fact that theN-terminal-most synthetic peptide is comprised of highly non-polar(hydrophobic) amino acids and is thus likely buried within the pilusfiber and inaccessible to antibody, this 15-mer peptide is anticipatedto serve as an internal negative control for the assays described here.Normal pediatric sera and naïve chinchilla sera will serve as negativeserum controls and middle ear lavage fluids recovered from a naïveanimal will be used as a negative control for effusions recovered duringNTHi infection of the middle ear.

Mapping Adhesin Binding Domains of PilA

In order to map the eukaryotic cell binding domains of PilA, competitiveELISA assays are conducted as well as evaluations of the ability of thesynthetic pilin peptides to inhibit NTHi binding to eukaryotic cells incell culture via confocal microscopy. For initial screening assays,relevant cukaryotic target cells are grown within 96-well microtiterdishes. Cells will be washed, then pre-incubated with synthetic pilinpeptides, rPilA or native NTHi pili [0.2 μg in PBS] to determine theirrelative ability to block binding of NTHi strain 86-028NP (grown underconditions known to promote pilin expression) to these eukaryotic cells.Relative adherence of NTHi will be determined using polyclonal antiseradirected against a homologous whole NTHi OMP preparation andHRP-conjugated protein A with color developed with tetramethylbenzidine(TMB). For these assays relevant epithelial target cells [i.e.chinchilla middle ear epithelial cells (CMEEs), normal humanbronchial/tracheal cells (NHuBr), human type II alveolar epithelial cellline (A549s)], a clinically irrelevant epithelial target cell to whichNTHi do not adhere (CHOs) as well as an endothelial target cells [humanumbilical vein endothelial cells (HUVECs)] will be used.

For those peptides that show inhibitory activity (typically the cut-offis at ≧15% inhibition of adherence relative to controls), anydose-dependence to the observed bacterial adherence blocking capabilityis determined. The interaction may be further evaluated by conductingadherence-blockade assays using a Transwell system wherein respiratorytract epithelial cells (CMEEs and NHuBrs) are grown at the air-fluidinterface. These cells are incubated with first synthetic peptides ofinterest (or appropriate controls, i.e. isolated OMP P5 and P2 aspositive and negative controls for NTHi surface proteins involved or notin adherence, respectively and rPilA) to attempt to block available Tfpreceptors, then they will be washed 5× with fresh growth medium followedby inoculation with ˜2-5×107 NTHi grown under conditions we know willpromote expression of Tfp. Cultures will be washed to removenon-adherent bacteria, then fixed with methanol on ice for 5 min, airdried, rinsed with PBS and the membranes removed from the Transwell andplaced on glass coverslips for imaging via confocal microscopy. Todetect adherent NTHi, chinchilla hyperimmune anti-NTHi OMP serum andFITC-Protein A will be used to document the interaction of NTHi with itsepithelial target cell, or conversely the blocking of this interactionby peptides that represent putative adhesin binding domains of PilA.

Choice of Immunogen

Based on the data acquired in above, immunogenic peptides are chosenbased on both relative immunodominance as well as ability to inhibitadherence of NTHi to respiratory epithelial target cells. Depending onthe biochemical and structural characteristics of the regions ofinterest, the peptides will be produced as either synthetic peptide(s)or recombinant peptide(s).

Immunogenicity and protective efficacy of the PilA immunogens isevaluated initially in the chinchilla animal model disclosed herein andin human trials.

Example Summary

The foregoing evidence indicates that NTHi express functional type IVpili on their surface. The proteins encoded by these genes are known tobe important for transformation competence in typeable H. influenzae andare contemplated herein to be important for biofilm formation by NTHi aswell. Collectively, these observations indicate that NTHi is likely toup-regulate expression of type IV pili in the nutrient restrictedenvironment of the human host. Thus, type IV pili represent an excellenttarget for a vaccine and/or for an antimicrobial strategy for pathogenicconditions caused by NTHi as well as H. influenzae strains a, b, c, eand f.

1. An isolated polynucleotide comprising a nucleotide sequence encodingan amino acid sequence of any one of: PilA polypeptide SEQ ID NO: 2,PilB polypeptide SEQ ID NO: 4, PilC polypeptide SEQ ID NO: 6, PilDpolypeptide SEQ ID NO: 8, ComA polypeptide SEQ ID NO: 10, ComBpolypeptide SEQ ID NO: 12, ComC polypeptide SEQ ID NO: 14, ComDpolypeptide SEQ ID NO: 16, ComE polypeptide SEQ ID NO: 18, ComFpolypeptide SEQ ID NO: 20, PilF polypeptide SEQ ID NO: 22, PilApolypeptide SEQ ID NO: 26, PilA polypeptide SEQ ID NO: 26, PilApolypeptide SEQ ID NO: 28, PilA polypeptide SEQ ID NO: 30, PilApolypeptide SEQ ID NO: 32, PilA polypeptide SEQ ID NO: 34, PilApolypeptide SEQ ID NO: 36, PilA polypeptide SEQ ID NO: 38, PilApolypeptide SEQ ID NO: 40, PilA polypeptide SEQ ID NO: 42, or pilApolypeptide SEQ ID NO:
 44. 2. An isolated polynucleotide comprising anucleotide sequence of any one of: pilA SEQ ID NO: 1, pilB SEQ ID NO: 3,pilC SEQ ID NO: 5, pilD SEQ ID NO: 7, comA SEQ ID NO: 9, comB SEQ ID NO:11, comC SEQ ID NO: 13, comD SEQ ID NO: 15, comE SEQ ID NO: 17, comF SEQID NO: 19, pilF SEQ ID NO: 21, pilA SEQ ID No: 25, pilA SEQ ID No: 27,pilA SEQ ID No: 29, pilA SEQ ID No: 31, pilA SEQ ID No: 33, pilA SEQ IDNo: 35, pilA SEQ ID No: 37, pilA SEQ ID No: 39, pilA SEQ ID No: 41 orpilA SEQ ID No:
 43. 3. A vector comprising a polynucleotide of claim 1.4. An isolated polypeptide comprising an amino acid sequence encoded bya nucleotide sequence of claim 1 or a fragment thereof.
 5. An isolatedpolypeptide comprising an amino acid sequence of any one of PilApolypeptide SEQ ID NO: 2, PilB polypeptide SEQ ID NO: 4, PilCpolypeptide SEQ ID NO: 6, PilD polypeptide SEQ ID NO: 8, ComApolypeptide SEQ ID NO: 10, ComB polypeptide SEQ ID NO: 12, ComCpolypeptide SEQ ID NO: 14, ComD polypeptide SEQ ID NO: 16, ComEpolypeptide SEQ ID NO: 18, ComF polypeptide SEQ ID NO: 20, PilFpolypeptide SEQ ID NO: 22, PilA polypeptide SEQ ID NO: 26, PilApolypeptide SEQ ID NO: 26, PilA polypeptide SEQ ID NO: 28, PilApolypeptide SEQ ID NO: 30, PilA polypeptide SEQ ID NO: 32, PilApolypeptide SEQ ID NO: 34, PilA polypeptide SEQ ID NO: 36, PilApolypeptide SEQ ID NO: 38, PilA polypeptide SEQ ID NO: 40, PilApolypeptide SEQ ID NO: 42, or pilA polypeptide SEQ ID NO: 44, or apeptide fragment thereof.
 6. A composition comprising a polypeptide orpeptide fragment of claim 5 and a pharmaceutically acceptable carrier.7. An antibody that specifically binds to a polypeptide or peptidefragment of claim
 5. 8. A composition comprising an antibody of claim 8and a pharmaceutically acceptable carrier.
 9. A method for detectingNTHi bacteria in a biological sample comprising (a) contacting apolynucleotide of claim 1 or a fragment thereof with a biologicalsample, and (b) detecting hybridization of the polynucleotide within thesample, wherein hybridization indicates the presence of NTHi bacteria.10. A method for detecting NTHi bacteria in a biological samplecomprising (a) contacting a polynucleotide of claim 2 or a fragmentthereof with a biological sample, and (b) detecting hybridization of thepolynucleotide within the sample, wherein hybridization indicates thepresence of NTHi bacteria.
 11. A method for detecting NTHi bacteria in abiological sample comprising: (a) contacting an antibody of claim 7 witha biological sample, and (b) detecting binding of the antibody withinthe sample, wherein binding indicates the presence of NTHi bacteria. 12.The method of claim 9 wherein the biological sample is selected from thegroup consisting of serum, sputum, ear fluid, blood, urine, lymphaticfluid, and cerebrospinal fluid.
 13. A method for eliciting an immuneresponse to NTHi bacteria comprising administering an immunogenic doseof one or more polypeptides or peptide fragments of claim 5 to a patientat risk of NTHi bacterial infection.
 14. A method for eliciting animmune response to NTHi bacteria comprising administering an immunogenicdose of one or more polynucleotides of claim 1 to a patient at risk ofNTHi bacterial infection.
 15. The method of claim 14 wherein thepolynucleotide is in a plasmid or viral vector.
 16. A method of treatingor preventing NTHi bacterial infection comprising administering amolecule that inhibits expression or activity of a polypeptide of claim5 to an patient in need thereof.
 17. The method of claim 16 wherein themolecule administered to the patient in need is an antisenseoligonucleotide.
 18. The method of claim 16 wherein the moleculeadministered to the patient in need is an antibody.
 19. The method ofclaim 16 wherein the molecule administered to the patient in need is asmall molecule.
 20. The method of claim 16 wherein the NTHi infection isin the middle ear.